Biochemical and theoretical approach to localization of metal-ion-binding sites in the actin primary structure

The number of Ca2+ ions bound at sites other than the single high-affinity site in CaCl2-induced polymers of rabbit skeletal muscle, chicken gizzard, and bovine aorta actin was determined. The polymer of skeletal muscle and aorta actin contained 4 mol Ca2+/mol, whereas gizzard actin only 3 mol weakly bound Ca2+/mol monomer. This difference correlates with the deletion in smooth muscle gamma-actin of one out of four NH2-terminal acidic residues typical of skeletal and smooth muscle alpha-actin isoforms, suggesting that this additional acidic residue in alpha-actins is involved in the weak binding of cations which is essential for polymerization. This experimental result, as well as a theoretical analysis of the actin primary structure, argue against the implication of the NH2-terminal acidic residues in the high-affinity site for divalent cation. The analysis of the actin primary structure aimed at identification of sequences resembling the known Ca2+-binding patterns has revealed the absence of an EF-hand Ca2+-binding site. The best match was obtained between the sequence of the 292-301 segment and that of Ca2+ site in lectins. However, in the light of experimental data discussed, it is more plausible that the actual high-affinity Ca2+ site in actin involves sequentially distant residues from the NH2- and COOH-terminal portions of the polypeptide chain.